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Abstract:

It is possible to stimulate the angiogenesis of an ischemic site declined
by diabetes and to recuperate ischemic disease by administering HGF
(hepatocyte growth factor) gene to the diabetic ischemic.

Claims:

1. A method for the treatment of diabetic lower limb ischemic disease in a
subject, comprising administering a therapeutically effective amount of a
hepatocyte growth factor (HGF) gene to the muscle of an ischemic site,
wherein expression of the HGF gene increases blood flow when compared to
an HGF-treated non-diabetic subject with ischemic disease, thereby
treating the diabetic lower limb ischemic disease.

2. The method according to claim 1, wherein the HGF gene is in the form of
a Sendai virus (HVJ)-liposome.

3. The method according to claim 1, wherein about 50 μg to about 5 mg
the HGF gene is administered to the subject, thereby treating the
diabetic ischemic disease.

4. The method of claim 1, wherein the HGF gene is administered as a naked
DNA.

5. The method of claim 1, wherein the HGF gene is administered as a naked
expression vector DNA.

6. The method of claim 1, wherein administration of the HGF gene increases
blood flow when compared to administration of an HGF-recombinant protein.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001]This is a continuation of U.S. patent application Ser. No.
12/080,715, filed Apr. 4, 2008, which is a continuation of U.S. patent
application Ser. No. 09/869,475, filed Oct. 10, 2001, which is the U.S.
National Stage of PCT Application No. PCT/JP00/07502, filed Oct. 26,
2000, which in turn claims priority to Japanese patent application Ser.
No. 11/309,984, filed Oct. 29, 1999. All applications are incorporated
herein in their entirety by reference.

TECHNICAL FIELD

[0002]The present invention relates to a gene therapy agent and gene
therapy method for diabetic ischemic disease utilizing a hepatocyte
growth factor (HGF) gene. More specifically, the present invention
relates to a method of gene therapy for diabetic ischemic disease which
comprises the noninvasive administration of therapeutic agents of
diabetic ischemic disease comprising an HGF gene as the effective
ingredient or HGF gene.

BACKGROUND ART

[0003]HGF is a protein that was first discovered as a strong growth factor
for mature hepatocytes and the gene encoding it has been cloned (Biochem.
Biophys. Res. Commun. 122, 1450 (1984); Proc. Natl. Acad. Sci. USA 83,
6489 (1986); FEBS Letter 22, 231 (1987); Nature 342, 440 (1989); Proc.
Natl. Acad. Sci. USA 87, 3200 (1991)). Afterwards, according to
researches, it has been revealed that HGF does not only work for repair
and regeneration of the damaged liver, as a hepatocyte regeneration
factor in vivo, but also has an angiogenic function and plays an
important role in treatment and prevention of ischemic disease and artery
disease (Symp. Soc. Exp. Biol. 47 cell behavior, 227-234 (1993); Proc.
Natl. Acad. Sci. USA 90, 1937-1941 (1993); Circulation 97, 381-390
(1998)). That is, it has been reported that upon administration of HGF to
the rabbit lower limb ischemic model, significant angiogenesis is
observed and improvement in blood flow, repression of blood pressure
decrease and improvement in ischemic symptoms take place. According to
these reports, it is believed today that HGF expresses and functions as
one of the angiogenic factors.

[0004]As stated above, HGF has various functions to begin with functions
as angiogenic factor, and many attempts have been made to use it as a
drug. However, the half life of HGF in blood arose as a problem. The half
life of HGF is as short as about 10 minutes, making it difficult to
maintain its concentration in blood. Thus, problems arose as to how to
deliver effective levels of HGF to the affected site.

[0005]Generally it is common knowledge that protein preparations are
mostly administered intravenously and concerning the case above of HGF
administration for the ischemic disease model, examples of intravenous
and intra-arterial administration are shown (Circulation 97, 381-390
(1998)). In spite of the fact that effectiveness against ischemic disease
or artery disease of intravenous or intra-arterial HGF administration to
such animal models are revealed, specific administration methods, doses
and so on effective for HGF are still under investigation. Particular
effective administration methods or doses and such for HGF proteins are
still to be determined, due to problems concerning its half life and
delivery to the affected site described above.

[0006]On the other hand, the rapid progress lately in molecular biology
has made it possible to activate cellular function by gene transfer
methods and various attempts have been made. Some trials have been made
for gene therapy of the heart region. There are some methods, like the
coronary diffusional infusion method and such, reported for gene transfer
methods but there is no case of gene transfer methods to the ischemic
site, particularly intramuscular infusion method to the skeletal muscle
showing effects on specific diabetic ischemic disease.

[0007]Further, it is known that angiogenesis hardly occurs and prognosis
is unfavorable in ischemic disease complicated with or caused by
diabetes. At present, it is not known whether HGF gene administration to
such diabetic ischemic disease is effective or not.

DISCLOSURE OF THE INVENTION

[0008]The object of this invention is to provide therapeutic agents and
treatment methods for diabetic ischemic disease that utilize the HGF
gene.

[0009]Inventors investigated to find out whether the HGF gene can be
adapted to diabetic ischemic disease and revealed that extremely
effective results are obtained by administering HGF gene directly to the
ischemic affected site. Specifically, relating to lower limb ischemic
disease, it was found out that effective results are obtained by
administering HGF gene to the lower limb layer. As mentioned above, it is
known that angiogenesis hardly occurs and prognosis is unfavorable in
ischemic disease complicated with or caused by diabetes. Therefore,
unlike mere ischemic disease, it had been unknown whether the HGF gene is
effective toward diabetic ischemic disease. This invention revealed the
effectiveness of the HGF gene for diabetic ischemic disease for the first
time.

[0010]Since this method is a non-invasive treatment, it has the advantage
that it is possible to administer the present gene repeatedly according
to the condition.

[0011]Thus, the outline of the present invention is as follows:

(1) a therapeutic agent for diabetic ischemic disease, which comprises
hepatocyte growth factor (HGF) as the effective ingredient;(2) the
therapeutic agent according to (1), used for administration to the
ischemic site;(3) the therapeutic agent according to (1) or (2), wherein
the diabetic ischemic disease is selected from the group consisting of
diabetic lower limb ischemic disease, diabetic ischemic neuropathy or
diabetic ischemic myocardial infarction;(4) the therapeutic agent
according to (3), wherein the diabetic ischemic disease is diabetic lower
limb ischemic disease;(5) the therapeutic agent according to any of (1)
to (4), used for administration into the muscle of the ischemic site;(6)
the therapeutic agent according to any of (1) to (5), wherein the HGF
gene is in the form of a Sendai virus (HVJ)-liposome;(7) the therapeutic
agent according to any of (1) to (6), which is to be administered
repeatedly as needed;(8) the therapeutic agent according to any of (1) to
(7), wherein the amount of HGF gene used is at least 50 μg;(9) a
method for the treatment of diabetic ischemic disease, which comprises
the transfer of the HGF gene into human;(10) the method according to (9),
wherein the HGF gene is administered to an ischemic site;(11) the method
according to (9) or (10), wherein the diabetic ischemic disease is
selected from the group consisting of diabetic lower limb ischemic
disease, diabetic ischemic neuropathy or diabetic ischemic myocardial
infarction;(12) the method according to (11), wherein the diabetic
ischemic disease is diabetic lower limb ischemic disease;(13) the method
according to any of (9) to (12), wherein the HGF gene is administered
into the muscle of ischemic site;(14) the method according to any of (9)
to (13), wherein the HGF gene is in the form of a Sendai virus
(HVJ)-liposome;(15) the method according to any of (9) to (14), wherein
the HGF gene is administered repeatedly as needed;(16) the method
according to any of (9) to (15), wherein the amount of HGF gene to be
administered is at least 50 μg;(17) use of the HGF gene for preparing
therapeutic agents for diabetic ischemic disease;(18) the use according
to (17), wherein the diabetic ischemic disease is selected from the group
consisting of diabetic lower limb ischemic disease, diabetic ischemic
neuropathy or diabetic ischemic myocardial infarction;(19) the use
according to (18), wherein the diabetic ischemic disease is diabetic
lower limb ischemic disease;(20) the use according to any of (17) to
(19), wherein the HGF gene is in the form of a Sendai virus
(HVJ)-liposome; and(21) the use according to any of (17) to (20), wherein
the amount of HGF gene to be used is at least 50 μg.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a graph showing changes in blood perfusion ratio over time
of the group of rats with diabetic lower limb ischemia in reference 1 and
of the control group, in which lower limb ischemia was induced in normal
rats.

[0013]FIG. 2 is a graph showing the internal HGF concentration in ischemic
muscle of the group of rats with diabetic lower limb ischemia in
reference 1 and of the control group, in which lower limb ischemia was
induced in normal rats.

[0014]FIG. 3 shows the blood perfusion ratio of the group of rats with
diabetic lower limb ischemia in Experiment 1, to which HGF gene was
administered or not, and of the control group, in which lower limb
ischemia was induced in normal rats.

[0015]FIG. 4 is a graph showing the result of a comparison of the number
of blood vessels of the group of rats with diabetic lower limb ischemia
in Experiment 1, to which HGF gene was administered or not, and of the
control group, in which lower limb ischemia was induced in normal rats by
ALP (alkaline phosphatase) staining of the skeletal muscle of the lower
limb ischemic site.

[0016]FIG. 5 is a graph showing the MMP-1 concentration in the culture
supernatant of the glucose added angioendothelial cell in reference 2, to
which HGF was added or not, and of the control group, to which no glucose
was added.

[0017]FIG. 6 is a graph showing the amount of mRNA of transcription factor
which is expressed in the angioendothelial cell, of the group of glucose
added angioendothelial cell in reference 3, to which HGF was added or
not, and of the control group to which no glucose was added.

BEST MODE FOR CARRYING OUT THE INVENTION

[0018]As used herein "HGF gene" means a gene that can express HGF (the HGF
protein). Specifically, cDNA of HGF described in Nature 342: 440 (1989);
Japanese Patent Publication No. 2777678; Biochem. Biophys. Res. Commun.
163: 967 (1989); and Biochem. Biophys. Res. Commun. 172: 321 (1990) and
so on integrated into suitable expression vectors (non-viral vector,
viral vector) described below are to be mentioned. The base sequence of
the cDNA encoding HGF gene of the present invention has been described in
the above literature and is also registered with databases such as
GENBANK. Thus, based on such sequence information, using a suitable DNA
portion as a PCR primer, it is possible to clone the cDNA of HGF, for
example, by performing a RT-PCR reaction on mRNA derived from the liver
or leukocytes. Such cloning can easily be performed by a person skilled
in the art according to a basic textbook, such as Molecular Cloning 2nd
Ed., Cold Spring Harbor Laboratory Press (1989).

[0019]The HGF genes of the present invention are not restricted to the
above mentioned genes but also include those genes that express proteins
with substantially the same function as HGF. That is, the following genes
fall under the category of the HGF gene of the present invention: 1) DNA
that hybridize to said cDNA under stringent conditions, and 2) DNA
encoding proteins having amino acid sequence in which 1 or more
(preferably a few) amino acids are substituted, deleted and/or added to
the protein encoded by said cDNA and which encodes proteins having the
function as HGF. Above DNAs of 1) and 2) can be readily obtained, for
example, by site-directed mutagenesis, PCR method, ordinal hybridization
method and so on. Such methods can be easily accomplished according to
the above basic textbook.

[0020]Subsequently, methods of gene transfer, dosage forms, doses and the
like for use in gene therapy of the present invention are explained.

[0021]The dosage form of a gene therapy agent comprising the above gene as
the effective ingredient to be administered to patients are roughly
classified into two groups: one is the case in which a nonviral vector is
used, and the other is in which a viral vector is used. Methods for
preparation and administration thereof are explained in detail in
experimental manuals (Supplement of Experimental Medicine, Basic
Technology in gene therapy, Yodosha (1996); Supplement of Experimental
Medicine, Experimental Methods in Gene Introduction and Expression
Analysis, Yodosha (1997); Handbook for Development and Research of Gene
Therapy, Japan Society of Gene Therapy ed., NTS (1999)). Specifics are
explained below.

[0022]A. Usage of a Nonviral Vector

[0023]Using a recombinant expression vector in which the gene of interest
has been integrated into a commonly used gene expression vector, may be
used to introduce the gene of interest into cells or tissue by the
following method etc.

[0025]Regarding methods of gene transfer into the tissue, a recombinant
expression vector may be incorporated into the cell by subjecting it to
any of the method, such as the method of gene transfer with internal type
liposome, method of gene introduction with electrostatic type liposome,
HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome
method), receptor-mediated gene introduction method, method of
introducing DNA molecules together with carriers (metal particles) by a
particle gun, method of directly introducing naked-DNA, method of
introduction with positively-charged polymers and the like.

[0026]Among them, HVJ-liposome is a fusion product prepared by enclosing
DNA into liposome made of lipid bilayer, which is fused to inactivated
Sendai virus (Hemagglutinating virus of Japan: HVJ). The HVJ-liposome
method is characterized by a very high fusing activity with the cell
membrane as compared to the conventional liposome method, and is a
preferred mode of introduction. For the method of preparing HVJ-liposome,
see the literature for details (Separate volume of; Experimental
Medicine, Basic Technology in gene therapy, Yodosha (1996); experimental
Methods in Gene Introduction and Expression Analysis, Yodosha (1997); J.
Clin. Invest. 93:1458-1464 (1994); Am. J. Physiol. 271: R1212-1220
(1996)) and the like, and experimental examples described below for
details. The HVJ-liposome method is exemplified by the method as
described in, for example, Molecular Medicine, 30, 1440-1448 (1993),
Experimental Medicine, 12, 1822-1826 (1994), and Protein, Nucleic Acid,
Enzyme, 42, 1806-1813 (1997), preferably that described in Circulation,
92 (Suppl. II), 479-482 (1995). In particular, the Z strain (available
from ATCC) is preferred as the HVJ strain, but other HVJ strains (for
example, ATCC VR-907 and ATCC VR-105) may also be used.

[0027]Furthermore, the method of directly introducing naked-DNA is the
most simple method among the methods described above, and in this regard
a preferred method of introduction.

[0028]Expression vectors as used herein may be any expression vectors so
long as they permit the in vivo expression of the gene of interest.
Examples include expression vectors such as pCAGGS (Gene 108:193-200
(1991)), pBK-CMV, pcDNA3.1, pZeoSV (INVITROGEN, STRATAGENE) and the like.

[0029]B. Usage of a Viral Vector

[0030]Representative methods that use viral vectors include those using
viral vectors such as recombinant adenovirus, retrovirus and the like.
More specifically, the gene of interest can be introduced into a DNA or
RNA virus such as detoxified retrovirus, adenovirus, adeno-associated
virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus,
Sendai virus, SV40, human immunodeficiency virus (HIV) and the like,
which is then infected to the cell to introduce the gene into the cell.

[0031]Among the above viral vectors, the efficiency of infection of
adenovirus is known to be much higher than that of other viral vectors.
In this regard, it is preferred to use an adenovirus vector system.

[0032]As methods of introducing a gene therapy agent into a patient, there
are in vivo methods, which permit direct introduction of the gene therapy
agent into the body, and ex vivo methods, in which certain cells are
removed from a human, wherein the gene therapy agent is introduced and
which are then returned into the body (Nikkei Science, April 1994 issue
pp. 20-24; Monthly Yakuji, 36(1):23-48 (1994); Supplement To Experimental
Medicine 12(15) (1994); Handbook for Development and Research of Gene
Therapy, NTS (1999)). According to the present invention, the in vivo
method is preferred.

[0033]Dosage forms may take various forms according to various
administration regimens described above (for example, liquids). When, for
example, an injection containing the gene as the effective ingredient is
to be used, said injection may be prepared by dissolving the effective
ingredients into a standard solvent (a buffer such as PBS, physiological
saline, sterile water, etc.). The injection liquid may then be
filter-sterilized with filter as needed, and then filled into sterilized
containers. Conventional carriers and so on may be added to the
injection. Liposomes, such as HVJ-liposome may take the form of
suspensions, frozen formulations, centrifugation-concentrated frozen
formulations and the like.

[0034]It is possible to use known factors having angiogenic functions,
additionally or alone besides the HGF gene used in this invention. For
example, it is reported that factors such as VEGF and FGF have an
angiogenic function and therefore such genes can be used. Further, growth
factors such as EGF are reported to repair cell damage in various tissues
and thus genes encoding them can be also used.

[0035]The diabetic ischemic disease herein includes diseases such as
diabetic lower limb ischemic disease, diabetic ischemic neuropathy and
diabetic ischemic cardiac infarction and so on, and the therapeutic agent
of this invention can be applied to any of these diseases. Moreover, the
therapeutic agent of this invention can be applied not only to patients
with critical diabetic ischemic disease but also to patients with
progressively mild symptoms.

[0036]Proper methods and sites for administration adequate for the disease
or symptom to be treated are selected for the gene therapy agent of this
invention. As to the administration methods, parenteral administration
methods are preferred. As a preferable administration site, the ischemic
site can be mentioned. "Ischemic site" herein refers to the site
including the affected site of ischemia and surrounding sites thereof.

[0037]Specifically, it is possible to administer into the blood vessel or
into the muscle of the ischemic site. However, administration into the
muscle of the ischemic site is preferred. In other words, administration
into the skeletal muscle of the lower limb ischemic site enables
stimulation of angiogenesis in the affected site of ischemia and
improvement of blood flow. Thereby, it enables recovery and normalization
of the function of the ischemic site. While in cardiopathy, such as
cardiac infarction, it is possible to gain similar effect by
administering into the cardiac muscle.

[0038]Examples of preferred administration methods include, for example,
administration by noninvasive catheter, noninvasive injector and so on.
Moreover, administration methods which utilize a noninvasive catheter,
noninvasive injector and such under the usage of echo can be mentioned.
As a method using noninvasive catheter, for example, injecting HGF genes
directly into the cardiac muscle from the ventricle inner space in a
cardiopathy can be indicated.

[0039]Application of the HGF gene of the present invention makes positive
treatment for patients with diabetic ischemic disease possible. For
example, it enables the recovery of function inpatients with critical
symptoms to whom no option, other than surgical excision of the affected
site, is left.

[0040]Dosage of the therapeutic agent of this invention varies depending
on the symptoms of the patient but HGF genes about 1 μg to about 50
mg, preferably about 10 μg to about 5 mg, more preferably about 50
μg to about 5 mg per adult patients can be defined.

[0041]The therapeutic agent of this invention is suited for administration
once every few days or once every few weeks and the frequency of
administration is appropriately selected depending on symptoms of
patients. According to the therapeutic treatment of the invention, genes
are administered noninvasively and, therefore, desired genes can be
administered as much as the condition demands.

[0042]The present invention will now be specifically explained with
reference to the following examples. It should be noted, however, that
the present invention is not limited by these examples in any way.

[0050]By surgically excising the femoral artery of one leg of the diabetic
rat (16 weeks old; 6 animals per group), to which diabetes was provoked
by interperitoneal administration of streptozotocin, ischemic state in
the lower limb site was produced.

[0053]After administration of the liposome preparation, the blood flow of
the lower limb was measured by laser Doppler imager (LDI) using laser
scatter analysis as the index of bypass circulation formation and effects
of improvement in blood flow. The average of colored histogram of
ischemic lower limb to that of the normal lower limb was taken as the
perfusion ratio.

[0054]The density of blood capillary in the lower limb ischemic site was
measured by alkaline phosphatase (ALP) staining, and the result of
diabetic lower limb ischemic rat group was compared to that of the
control lower limb ischemic rat group. Alternatively, comparison between
the groups to which HGF gene was administered and to which no HGF gene
was administered was made.

[0055]Reference 1

[0056]HGF Expression State in the Lower Limb Ischemic Site of the Diabetic
Rat

[0057]Ischemic state in the lower limb site was produced by surgical
excision of the femoral artery of one leg of the diabetic rats (16 weeks
old; 6 animals per group), to which diabetes was provoked by
interperitoneal administration of streptozotocin, and of normal rats (6
weeks old; 6 animals per group) as the control group.

[0058]After one week, the perfusion ratio of the ischemic site was
measured by laser Doppler imager. The perfusion ratio of the ischemic
site or the diabetic lower limb ischemic rat was much lower than that of
the control lower limb ischemic rat (see FIG. 1).

[0059]The perfusion ratio of the lower limb was measured again 3 weeks and
5 weeks later, and the same results were obtained. That is, the perfusion
ratio of the lower limb of the diabetic lower limb ischemic rat was much
lower than that of the control lower limb rat (see FIG. 1).

[0060]The internal HGF concentration in the muscles was significantly
lower in the muscles of the ischemic site of the diabetic lower limb
ischemic rat than that of the control lower limb ischemic rat. This
result indicates that angiogenesis in diabetes is poor due to the
decrease of internal HGF in the muscles. Therefore, angiogenesis hardly
occurs in diabetic lower limb ischemic rat and bypass circulation does
not develop (see FIG. 2).

[0063]Ischemic state in the lower limb site was produced by surgical
excision of the femoral artery of one leg of the normal rats (16 weeks
old; 6 animals per group) and that of diabetic rats (16 weeks old; 6
animals per group), to which diabetes was provoked by interperitoneal
administration of streptozotocin. After surgical excision of the femoral
artery, infusion of HVJ-liposome preparation containing HGF gene (50
μg) was injected into the muscle of the lower limb ischemic site.

[0064]After 3 weeks, the perfusion ratio of the ischemic site was measured
by laser Doppler imager. The perfusion ratio of the ischemic site of the
diabetic lower limb ischemic rat, to which HGF gene was administered,
showed significant increase compared to that of the control lower limb
ischemic rat or that of the diabetic lower limb ischemic rat above with
no administration.

[0065]Taking perfusion ration of the control lower limb ischemic rat as
100%, that of the HGF gene untreated diabetic lower limb ischemic rat was
67% and that of the HGF administered diabetic lower limb ischemic rat was
129%. The results are shown in FIG. 3 (see FIG. 1).

[0068]Diabetic lower limb ischemic rat and control lower limb ischemic rat
treated as above were prepared and subjected to HGF gene therapy. 5 weeks
later, the skeletal muscle of the lower limb ischemic site was taken from
each animal, subjected to ALP staining and the blood vessel count per
unit area was compared. The blood vessel count of HGF gene untreated
diabetic lower limb ischemic rat was significantly smaller as that of the
control lower limb ischemic rat, and that of the HGF administered
diabetic lower limb ischemic rat was significantly increased. The results
are shown in FIG. 4.

[0069]Reference 2

[0070]Influence of Glucose Concentration and HGF Addition Against MMP-1
Production of the Angioendothelial Cell

[0071]The angioendothelial cells (derived from human aorta) were cultured
in three types of serum free medium containing glucose at a concentration
of 0, 25 mM and 50 mM, respectively. After 24 hours of cultivation, the
MMP-1 concentration in the supernatant of the culture media was measured.

[0072]Each sample was compared to that in which 100 ng/ml of HGF was added
30 minutes before glucose addition.

[0073]The MMP-1 concentration of the supernatant decreased significantly
depending on the glucose concentration, and it was shown that HGF
treatment inhibited the decrease of MMP-1 and, in turn, induced its
increase. The results are shown in FIG. 5.

[0076]Cultivation of angioendothelial cell was conducted as in reference
1, and expression of mRNA of the transcription factor ETS-1 in the cell
was detected. Taking the mRNA of ETS-1 in the control endothelial cell as
100%, that of the HGF untreated angioendothelial cell decreased in a
glucose dependent manner. On the other hand, HGF treated angioendothelial
cells expressed the mRNA of ETS-1 at the same or more level compared to
that of the control group (P<0.01). The results are shown in FIG. 6.

[0077]As described above, angioendothelial cells under high glucose
concentration show a decrease in MMP-1 expression, which is a matrix
cleaving enzyme essential for angiogenesis, and show a decrease in the
expression of mRNA of the transcription factor ETS-1, which is expressed
and increases during angiogenesis.

[0078]Consequently, it was revealed that angiogenesis hardly occurs under
high glucose condition. On the other hand, it was shown that by adding
HGF to the angioendothelial cell under high glucose condition, MMP-1
expression and mRNA of ETS-1 expression increases significantly. Thus, it
was revealed that HGF makes angiogenesis easier.

INDUSTRIAL APPLICABILITY

[0079]The therapeutic agent for diabetic ischemic disease containing an
HGF gene as the effective ingredient improves poor angiogenesis specific
to the affected site of diabetic ischemia with decrease in HGF expression
and shows significant angiogenic effect. Therefore, it enables the
improvement of the condition by increasing the blood flow in the affected
site of ischemia. Moreover, the therapeutic agent of this invention can
be administered once or more, depending on the symptoms of the patient,
thereby stimulating angiogenesis. Therefore, according to these effects,
the therapeutic agent of this invention makes it possible to treat
diabetic lower limb ischemic disease, diabetic ischemic neuropathy and
diabetic cardiac infarction.